Bruton's Tyrosine Kinase Inhibitor Research Articles

A multicenter study of venetoclax-based treatment for patients with Richter transformation of chronic lymphocytic leukemia

AbstractPatients with chronic lymphocytic leukemia (CLL) who develop Richter transformation (RT) have a poor prognosis when treated with chemoimmunotherapy regimens used for de novo diffuse large B-cell lymphoma. Venetoclax, a BCL2 inhibitor, has single-agent efficacy in patients with RT and is potentially synergistic with chemoimmunotherapy. In this multicenter, retrospective study, we evaluated 62 patients with RT who received venetoclax-based treatment outside of a clinical trial, in combination with a Bruton tyrosine kinase inhibitor (BTKi; n=28), rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) (n=13), or intensive chemoimmunotherapy other than R-CHOP (n=21). The best overall and complete response rates were 36%/25%, 54%/46%, and 52%/38%, respectively. The median progression-free and overall survival estimates for the same treatment groups were 4.9/14.3 months, 14.9 months/not reached, and 3.3/9 months, respectively. CLL with del(17p) was associated with a lower complete response rate in the total cohort (odds ratio [OR] 0.15; 95% confidence interval [CI] 0.04-0.6; p=0.01) and venetoclax-naïve subgroup (OR 0.13; 95%CI 0.02-0.66; p=0.01). TP53 mutated CLL was associated with a lower complete response rate (OR 0.15; 95%CI 0.03-0.74; p=0.02) and shorter progression-free survival (hazard ratio 3.1; 95%CI 1.21-7.95; p=0.02) only in venetoclax-naïve subgroup. No other clinical or baseline characteristics, including prior venetoclax treatment for CLL, showed statistically significant association with outcomes. Grade 3-4 neutropenia and thrombocytopenia events were most frequent with intensive chemoimmunotherapy + venetoclax; grade 3-4 infection rates were similar across treatment groups. In this difficult-to-treat RT patient population, venetoclax-based combination regimens achieved high response rates, with durable remission and survival observed in a subset of patients.

Targeting the B cell receptor signaling pathway in chronic lymphocytic leukemia

Aberrant signal transduction through the B cell receptor (BCR) plays a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). BCR-dependent signaling is necessary for the growth and survival of neoplastic cells, making inhibition of down-stream pathways a logical therapeutic strategy. Indeed, selective inhibitors against Bruton's tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) have been shown to induce high rates of response in CLL and other B cell lymphomas. In particular, the development of BTK inhibitors revolutionized the treatment approach to CLL, demonstrating long-term efficacy. While BTK inhibitors are widely used for multiple lines of treatment, PI3K inhibitors are much less commonly utilized, mainly due to toxicities. CLL remains an incurable disease and effective treatment options after relapse or development of TKI resistance are greatly needed. This review provides an overview of BCR signaling, a summary of the current therapeutic landscape, and a discussion of the ongoing trials targeting BCR-associated kinases.

Research Progress of Targeted Therapy for Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma --Review

Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) is a relatively inert B lymphocyte proliferative disease. In recent years with the launch of new drugs, chemotherapy has been gradually replaced by targeted therapy, which significantly prolongs the survival of patients and reduces the side effects of treatment. At present, BTK inhibitors, PI3K inhibitors, spleen tyrosine kinase (SYK) inhibitors and BCL-2 inhibitors are the most studied targeted therapeutic drugs for CLL/SLL. This article reviews the research progress of different types of targeted therapeutic drugs in the treatment of CLL/SLL.

Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma

The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both invitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.

Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy for Richter Transformation: An International, Multicenter, Retrospective Study.

Outcomes for Richter transformation (RT) are poor with current therapies. The efficacy and safety of anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T) for RT are not established. We performed an international multicenter retrospective study of patients with RT who received CAR-T. Patient, disease, and treatment characteristics were summarized using descriptive statistics, and modeling analyses were used to determine association with progression-free survival (PFS) and overall survival (OS). PFS and OS were estimated from the date of CAR-T infusion. Sixty-nine patients were identified. The median age at CAR-T infusion was 64 years (range, 27-80). Patients had a median of four (range, 1-15) previous lines of therapy for CLL and/or RT, including previous Bruton tyrosine kinase inhibitor and/or BCL2 inhibitor therapy in 58 (84%) patients. The CAR-T product administered was axicabtagene ciloleucel in 44 patients (64%), tisagenlecleucel in 17 patients (25%), lisocabtagene maraleucel in seven patients (10%), and brexucabtagene autoleucel in one patient (1%). Eleven patients (16%) and 25 patients (37%) experienced grade ≥3 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, respectively. The overall response rate was 63%, with 46% attaining a complete response (CR). After a median follow-up of 24 months, the median PFS was 4.7 months (95% CI, 2.0 to 6.9); the 2-year PFS was 29% (95% CI, 18 to 41). The median OS was 8.5 months (95% CI, 5.1 to 25.4); the 2-year OS was 38% (95% CI, 26 to 50). The median duration of response was 27.6 months (95% CI, 14.5 to not reached) for patients achieving CR. CAR-T demonstrates clinical efficacy for patients with RT.

Computational Investigation of the Covalent Inhibition Mechanism of Bruton's Tyrosine Kinase by Ibrutinib.

Covalent inhibitors represent a promising class of therapeutic compounds. Nonetheless, rationally designing covalent inhibitors to achieve a right balance between selectivity and reactivity remains extremely challenging. To better understand the covalent binding mechanism, a computational study is carried out using the irreversible covalent inhibitor of Bruton tyrosine kinase (BTK) ibrutinib as an example. A multi-μs classical molecular dynamics trajectory of the unlinked inhibitor is generated to explore the fluctuations of the compound associated with the kinase binding pocket. Then, the reaction pathway leading to the formation of the covalent bond with the cysteine residue at position 481 via a Michael addition is determined using the string method in collective variables on the basis of hybrid quantum mechanical-molecular mechanical (QM/MM) simulations. The reaction pathway shows a strong correlation between the covalent bond formation and the protonation/deprotonation events taking place sequentially in the covalent inhibition reaction, consistent with a 3-step reaction with transient thiolate and enolates intermediate states. Two possible atomistic mechanisms affecting deprotonation/protonation events from the thiolate to the enolate intermediate were observed: a highly correlated direct pathway involving proton transfer to the Cα of the acrylamide warhead from the cysteine involving one or a few water molecules and a more indirect pathway involving a long-lived enolate intermediate state following the escape of the proton to the bulk solution. The results are compared with experiments by simulating the long-time kinetics of the reaction using kinetic modeling.

A prospective study of zanubrutinib, a Bruton tyrosine kinase inhibitor, in relapsed/refractory idiopathic multicentric Castleman disease.

Relapsed and refractory (R/R) idiopathic multicentric Castleman disease (iMCD) is a clinical challenge with no standard treatment. In this preliminary clinical trial, we investigated the efficacy and safety profiles of a Bruton tyrosine kinase inhibitor (BTKi), zanubrutinib, in patients with R/R iMCD. The primary endpoint was theoverall response rate at Week 12according to theCastleman Disease Collaborative Network (CDCN) response criteria. The trial was terminated early due to a lack of treatment response in the first enrolled 5 patients. Although 3 patients achieved symptomatic response, none of the 5 patients had an overall response by Week 12. One patient had progressive disease and the other 4 had stable disease. The study drug was well tolerated without grade 2 or higher adverse events. Our findings suggest that BTKi therapy is not effective for iMCD, and further attempts at single-agent therapy with zanubrutinib or other BTKis for iMCD should be consideredwith caution and probably avoided. This trial was registered at www.clinialtrials.gov as #NCT04743687.

Bruton Tyrosine Kinase Inhibition: an Effective Strategy to Manage Waldenström Macroglobulinemia.

The treatment of Waldenström macroglobulinemia (WM) has evolved over the past decade. With the seminal discoveries of MYD88 and CXCR warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) mutations in WM cells, our understanding of the disease biology and treatment has improved. The development of a new class of agents, Bruton tyrosine kinase inhibitors (BTKi), has substantially impacted the treatment paradigm of WM. Herein, we review the current and emerging BTKi and the evidence for their use in WM. Clinical trials have established the role of covalent BTKi in the treatment of WM. Their efficacy is compromised among patients who harbor CXCR4WHIM mutation or MYD88WT genotype. The development of BTKC481 mutation-mediated resistance to covalent BTKi may lead to disease refractoriness. Novel, non-covalent, next-generation BTKi are emerging, and preliminary results of the early phase clinical trials show promising activity in WM, even among patients refractory to a covalent BTKi. Covalent BTK inhibitors have demonstrated meaningful outcomes in treatment-naïve (TN) and relapsed refractory (R/R) WM, particularly among those harboring the MYD88L265P mutation. The next-generation BTKi demonstrate improved selectivity, resulting in a more favorable toxicity profile. In WM, BTKi are administered until progression or the development of intolerable toxicity. Consequently, the potential for acquired resistance, the emergence of cumulative toxicities, and treatment-related financial burden are critical challenges associated with the continuous therapy approach. By circumventing BTK C481 mutations that alter the binding site to covalent BTKi, the non-covalent BTKi serve as alternative agents in the event of acquired resistance. Head-to-head comparative trials with the conventional chemoimmunotherapies are lacking. The findings of the RAINBOW trial (NCT046152), comparing the dexamethasone, rituximab, and cyclophosphamide (DRC) regimen to the first-generation, ibrutinib are awaited, but more studies are needed to draw definitive conclusions on the comparative efficacy of chemoimmunotherapy and BTKi. Complete response is elusive with BTKi, and combination regimens to improve upon the efficacy and limit the treatment duration are also under evaluation in WM.

Bruton tyrosine kinase inhibitors in multiple sclerosis: evidence and expectations.

Despite availability of high-efficacy therapies for multiple sclerosis (MS), many patients experience significant disability worsening due to limited effects of currently available drugs on central nervous system (CNS)-compartmentalized inflammation. Bruton tyrosine kinase (BTK) is an intracellular signaling molecule involved in regulation of maturation, survival, migration, and activation of B cells and microglia, which are central players in the immunopathogenesis of progressive MS. Therefore, CNS-penetrant BTK inhibitors may better prevent disease progression by targeting immune cells on both sides of the blood-brain barrier. This review gives an overview on the preliminary results of clinical trials. Currently, the efficacy and safety of six BTK inhibitors are being evaluated in clinical trials in patients with relapsing and progressive MS. Evobrutinib, tolebrutinib and fenebrutinib have shown efficacy and safety in relapsing MS in phase 2 studies, and evobrutinib and tolebrutinib in their extension studies up to 3-5 years. However, evobrutinib failed to distinguish itself from the comparator drug teriflunomide in reduction of relapse rate (primary end point) in two phase 3 studies in relapsing MS. Inhibition of BTK has emerged as a promising therapeutic approach to target the CNS-compartmentalized inflammation. Results from phase 3 clinical trials will shed light on differences in efficacy and safety of BTK inhibitors and its potential role in the future MS landscape.

Functional human skin explants as tools for assessing mast cell activation and inhibition.

Mast cells are activated through a variety of different receptors to release preformed granules and mediators synthesized de novo. However, the physiology and function of mast cells are not fully understood. Traditional studies of mast cell activation in humans have utilized cultures of tissue-derived mast cells including CD34+ progenitor cells or well-characterized commercially available cell lines. One limitation of these methods is that mast cells are no longer in a natural state. Therefore, their applicability to human skin disorders may be limited. Human skin explant models have been utilized to investigate the short-term effects of cell mediators, drugs, and irritants on skin while avoiding the ethical concerns surrounding in vivo stimulation studies with non-approved agents. Nonetheless, few studies have utilized intact human tissue to study mast cell degranulation. This "Methods" paper describes the development and application of an intact skin explant model to study human mast cell activation. In this manuscript, we share our protocol for setting up ex vivo human skin explants and describe the results of stimulation experiments and techniques to minimize trauma-induced histamine release. Skin explants were generated using de-identified, full-thickness, non-diseased skin specimens from plastic and reconstructive surgeries. Results were reproducible and demonstrated FcɛRI- and MRGPRX2-induced mediator release which was inhibited with the use of a BTK inhibitor and QWF, respectively. Thus, this explant model provides a quick and accessible method of assessing human skin mast cell activation and inhibition.

Abstract 929: Characterizing first- & second-generation follow on drugs: Clinical trial & development timelines of Bruton tyrosine kinase (BTK) inhibitors

Abstract Once a promising new cancer therapy emerges, other manufacturers often develop drugs with the same molecular target. Some follow on drugs offer meaningful benefit via improved efficacy or reduced toxicity, but others offer minimal benefit. We hypothesized there are two cohorts of follow on drugs: those developed concurrently before efficacy & safety of the therapy is established (first generation) & those developed as follow ons after that point (second generation). We analyzed BTK inhibitor (BTKi) development to evaluate patterns of concurrent & follow on development. Methods BTKis from 1996-2023 were identified in Pharmaprojects (Citeline, NY). We collected drug development information including indications pursued, development phase reached, & outcome (ongoing development, discontinued development, or completed development with FDA approval). We constructed timelines including the first publicized report of the drug, Phase 2 initiation & date of development discontinuation or FDA approval. Results We identified 19 BTKis targeting CLL, of which 6 (32%) were concurrent and 13 (68%) were follow-ons relative to ibrutinib’s 2014 approval. A cluster of new agents began development immediately after ibrutinib approval, suggesting two distinct groups in this case. We identified 10 BTKis for MCL, of which only 2 (20%) were concurrently developed drugs, and 8 (80%) were follow on, based on ibrutinib’s 2013 approval for MCL, with similar clustering to CLL. Conclusions Among BTKis, market entry progressed in two phases. In a first phase, a smaller group of sponsors attempted to demonstrate safety & efficacy of the novel class. After proof of safety & efficacy, lowering risks for later competitors, a follow on phase of development began in which multiple sponsors sought to enter the market. Knowledge of which drugs belong to which class can aid with evaluation of trial design as part of regulatory approval decisions & with drug price negotiation. Bruton Tyrosine Kinase inhibitors in Clinical Stage Development in Chronic Lymphocytic Leukemia Drug Sponsor Entry Date Date of approval/discontinuation Outcome Ibrutinib AbbVie/J&J 12/12/2007 02/13/2014 Approved (first in class) Spebrutinib Bristol Myers Squibb 12/12/2008 01/16/2014 Discontinued Vecabrutinib Biogen/Viracta 04/07/2011 03/23/2016 Discontinued Tirabrutinib Gilead/Ono 01/14/2012 09/19/2023 Discontinued Fenebrutinib Roche 11/26/2013 Unknown Discontinued Pirtobrutinib Eli Lilly 12/27/2013 08/08/2023 Approved Acalabrutinib Acerta/AstraZeneca 01/01/2014 06/11/2019 Approved Luxeptinib Apstose Biosciences 08/19/2014 - Ongoing Zanubrutinib BeiGene 08/27/2014 01/14/2022 Approved Nemtabrutinib Merck 05/06/2016 - Ongoing Orelabrutinib Biogen, Beijing Innocare 06/20/2017 - Ongoing AS-1763 Carna Biosciences 11/24/2017 - Ongoing JNJ-624264681 J&J 08/03/2018 - Ongoing HMPL-760 Hutchmed 06/10/2020 - Ongoing Citation Format: Ashwin Varma, William B. Feldman, Aaron S. Kesselheim, Edward R. Cliff. Characterizing first- & second-generation follow on drugs: Clinical trial & development timelines of Bruton tyrosine kinase (BTK) inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 929.

Abstract 5966: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies

Abstract CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer cell growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. Specifically, we show that brief treatment with PRT2527 (6h) potently induces apoptosis and inhibits proliferation in DLBCL, MCL and CLL cell lines. We also show that the addition of BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) potentiates the apoptosis response induced by PRT2527 in vitro and leads to more robust and durable responses including complete tumor regressions in DLBCL CDX and PDX models in vivo. In all studies, the combination of PRT2527 with BTKi or BCL2i is well tolerated. Mechanistically, we show that BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency in TMD-8 cells. Concurrent depletion of MCL1 and BFL1 with PRT2527 leads to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In similar fashion, Venetoclax-driven BCL2 inhibition also potentiates the anti-tumor activity of PRT2527. Consistent with these findings in vitro, we observe reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrate potent induction of apoptosis in peripheral blood mononuclear cells isolated from patients with both treatment-naïve and relapse/refractory CLL following brief treatment with PRT2527 (6h). Co-treatment with BTKi or BCL2i further enhances the apoptotic effect of PRT2527, similarly suggesting combinatorial benefit. Additionally, we characterize the activity of PRT2527 in models of BTKi relapsed/refractory MCL where increased CDK9 dependency has previously been described. In an Ibrutinib-resistant Mino CDX model, we observe increased tumor growth inhibition following treatment with PRT2527 compared to tumors derived from parental Mino cells, suggesting PRT2527 is efficacious in BTKi-resistant MCL. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory lymphoid malignancies. Citation Format: Norman Fultang, Ashley M. Schwab, Sophia McAneny-Droz, Diane Heiser, Peggy Scherle, Neha Bhagwat. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5966.

Abstract 4669: Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations

Abstract Background: Differences in profiles of acquired mutations of resistance to BTKis might have implications for treatment sequencing. We evaluated preclinical activity of BTKis (ibrutinib [Ibr], acalabrutinib [Acala], zanubrutinib [Zanu], and pirtobrutinib [Pirto]) against multiple clinically relevant BTK mutations and report on the clinical impact of BTK C481S in patients (pts) treated with Ibr. Methods: Inhibition of kinase activity by BTKis was evaluated in a cell-free kinase activity assay (Nanosyn). Cell-killing activity on CRISPR-generated BTK mutant knock-in TMD8 cells was assessed after 3 days of BTKi treatment. Time from first detection of C481S mutation to progressive disease (PD) was evaluated in pts treated with Ibr with previously untreated (n=247) or relapsed/refractory (R/R; n=172) CLL using pooled data from 5 clinical trials. Results: In vitro kinase activity was highest for Ibr compared with Acala or Zanu against C481S (Ibr IC50: 26.3 nM; Zanu: 84.8 nM; Acala: 338 nM) and T474I (Ibr: 1.2 nM; Zanu: 4.4 nM; Acala: 43.6 nM). Ibr maintained cell-killing activity against all tested BTK mutants, whereas limited activity was seen with Acala against C481S and A428D; with Zanu against C481S, L528W, and A428D; and with Pirto against T474I, L528W, V416L, and A428D (Table). In pts treated with Ibr, C481S was detected prior to clinical PD in 3 previously untreated pts and in 26 pts with R/R CLL; median (range) time from first detection of C481S to PD in these pts was 17.0 mo (11.2-34.3) and 10.0 mo (2.6-35.7), respectively. Conclusion: This study demonstrates activity of Ibr against all evaluated BTK variants, which are associated with resistance to covalent and noncovalent BTKis. Clinical data shows that pts who develop C481S mutations continue to derive benefit from Ibr for several months before PD. Together, these data suggest a role for Ibr in the treatment of pts with a broad spectrum of BTK variants. Table. Cell-killing activity of BTKis against wild type and mutant BTK EC50 (nM) BTK Wild type C481S T474I L528W V416L A428D Ibr 0.6 414.0 1.9 181.4 0.5 205.3 Acala 3.9 2973.8 43.3 3.5 106.3 3000.0 Zanu 0.7 2322.0 11.7 3000.0 0.6 1726.0 Pirto 8.2 12.6 2727.5 3000.0 1898.0 3000.0 Citation Format: Matthew S. Davids, Inhye E. Ahn, Jacob J. Riehm, Melih Acar, Vincent Girardi, Sima Patel, Tarikere Gururaja, Jennifer Woyach, Lloyd T. Lam, Edith Szafer-Glusman. Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4669.

Abstract 1960: CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations

Abstract Bruton’s Tyrosine Kinase (BTK) inhibitor drugs are a mainstay in the treatment of CLL, WM and MCL. They are not curative because of the emergence of BTK inhibitor resistance mutations in as much as 67% of relapsed patients (in CLL) [1]. Frequently observed mutations include BTKC481S, which abrogates covalent binding of irreversible drugs such as acalabrutinib, ibrutinib and zanubrutinib, and BTKT474I and BTKV416L which both abrogate binding of the non-covalent inhibitor drug pirtobrutinib [1, 2]. To combat resistance, third generation BTK inhibitors are needed, which must 1. bind to unique regions in the tumor driving kinases that are not marred by resistance mutations, 2. have sufficient potency for sustained inhibition of BTK signaling, and 3. be sufficiently selective to afford efficacious doses and suitable for a combination regimen. We have developed a platform called Energetically Privileged Ligands (EPriLs), based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket. Their unique binding mode involves connecting the N-terminal and C-terminal lobes of the general kinase fold in a continuous β-sheet, in this way avoiding hotspots for kinase inhibitor resistance mutations. The potent binding of the macrocycle ensures a very long residence time. Using the EPriL platform, we have developed CFON-026, a next generation non-covalent BTK inhibitor with a covalent-like residence time as demonstrated by surface plasmon resonance (SPR) and cellular washout experiments. The biochemical binding KD to BTKWT, determined by SPR, is 0.14 nM. CFON-026 binds mutants with KDs of 0.4 nM (BTKC481S) and and 1.8 nM (BTKT474I). X-ray crystal structures of CFON-026 bound to BTKWT and BTKT474I confirm the unique binding mode and water network around the active site. CFON-026 inhibits the growth of the BTK dependent cell lines TMD8 and Rec-1 with IC50s of 0.45 nM and 1.7 nM, respectively. CFON-026 is very active on TMD8 variants containing endogenous BTK inhibitor resistance mutations (BTKC481S, BTKV416L, BTKT474I and BTKL528W, IC50 range 1-100 nM). CFON-026 has favorable metabolic stability, pharmacokinetic properties, and safety characteristics, and induces complete tumor regression in a TMD8 xenograft model. Since BTK resistance mutations can be detected diagnostically 12-18 months before clinical relapse, CFON-026 could be applied as combination partner to prolong progression free survival on first line BTK inhibitor therapy. Experiments with mixed populations of BTK mutant cell lines, to model a heterogenous tumor, show that CFON-026 can deepen the efficacy of ibrutinib, acalabrutinib and zanubrutinib. In conclusion, CFON-026 is a novel EPriL-based non-covalent BTK inhibitor that could enhance and prolong responses of existing covalent BTK inhibitor therapy. [1] Woyach et al., J Clin Oncol (2017). [2] Wang et al., N. Eng J Med (2022). Citation Format: Jos de Man, Michelle Muller, Joost C.M. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G.T.H. van Mil, Winfried R. Mulder, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1960.

Abstract 605: ABBV-101, a highly potent and selective clinical stage Bruton tyrosine kinase degrader, overcomes BTK mutation-induced resistance to BTK inhibitors

Abstract Bruton tyrosine kinase (BTK), a clinically validated target for various B-cell malignancies, plays a critical role in regulating cell growth, adhesion, and homing to lymphoid tissues that provide a microenvironment favorable to cancer cells. Despite the success of approved covalent BTK inhibitors in diseases such as CLL, most patients do not achieve complete response with monotherapy and eventually relapse, often via the emergence of the BTK-C481S mutation. Although second-generation reversible BTK inhibitors may temporarily overcome the C481S mutation, novel BTK mutations associated with resistance emerged in the clinic recently. Additionally, covalent BTK inhibitors have not shown sufficient differentiation from standard-of-care (SoC) in B-cell malignancies, such as diffuse large B cell lymphoma (DLBCL) to warrant approval to date. To address these unmet needs, a next-generation orally bioavailable BTK degrader ABBV-101 has been developed. Unlike BTK inhibitors that solely bind to and impede the catalytic domain of BTK, ABBV-101 eliminates the protein in a highly selective fashion. This degradation mechanism targets both the catalytic and scaffolding functions of BTK, and thus may enable deeper and more durable responses in patients with B-cell malignancies. Cellular activity of ABBV-101 was determined in a panel of DLBCL cell lines. We show that most non-germinal center B cell DLBCL (non-GCB-DLBCL) cell lines are sensitive to ABBV-101. To determine whether ABBV-101 could overcome BTK mutations-induced resistance, we engineered human DLBCL cell line TMD8 using CRISPR technology to express these BTK mutations and tested for sensitivity to ABBV-101 and BTK inhibitors. We show that ABBV-101 demonstrates similar potent activity against all BTK mutations associated with resistance to both covalent and reversible BTK inhibitors. The modulation of downstream pathway by ABBV-101 and how it differentiates from other BTK inhibitors will be presented. Further, ABBV-101 induces complete tumor regression in the BTK-C481S TMD8 DLBCL xenograft model. A spontaneous mouse CLL model carrying BTK-C481S mutation was developed through crossing BTK-C481S knock-in mice with the Emu-TCL1 transgenic mice in the C57BL/6 background. ABBV-101 completely inhibits BTK-C481S Emu-TCL1 CLL burden increase in the blood compartment and reduces CLL burden in the spleen and lymph nodes, whereas covalent BTK inhibitors show no activity. Finally, combination with a BCL-2 inhibitor enhances the efficacy of ABBV-101 in CLL and DLBCL models. Taken together, our results suggest that ABBV-101 has potent in vitro and in vivo efficacy. ABBV-101 is currently in phase 1 clinical trial for a variety of B-cell malignancies. ClinicalTrials.gov identifier: NCT05753501 Citation Format: Chin Pan, Jacob Riehm, Tarikere L. Gururaja, Haiyan Li, Henry Nguyen, Yongjiao Zhai, Xiaoping Xie, Rong-Xian Ding, Rebecca Matthew, Hana Y. Hoh, Zhaozhong J. Jia, Bo Liu, Cassandra P. Shu, Claudina A. Stevenson, Christine Will, Federico Innocenti, Wissam Assaily, Lloyd T. Lam, Alexey Rivkin. ABBV-101, a highly potent and selective clinical stage Bruton tyrosine kinase degrader, overcomes BTK mutation-induced resistance to BTK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 605.

Abstract 6509: GT0486, a novel mTORC1/2 dual inhibitor, exhibits synergistic antitumor efficacy in combination with BTK inhibitors

Abstract Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is the central signal molecule in regulating cell growth, metabolism, angiogenesis, and survival. The first generation of mTOR inhibitors specifically inhibit mTORC1 but are insensitive to mTORC2 and cannot eliminate the downstream signaling cascade in cancer. GT0486, a second-generation mTORC1/2 dual inhibitor, can selectively suppress mTOR kinase activity while has no inhibitory effect on other kinase receptors. We investigate the anti-tumor efficacy of GT0486 in a variety of tumor cells and xenograft animal models in vitro and in vivo. The combination effects of GT0486 and BTKi (BTK inhibitor) were also evaluated. Compared with other mTOR inhibitors (GDC-0349, AZD-2014, Rapamycin, GDC-0941, and CC-223), GT0486 exhibits the stronger inhibitory activity on human tumor cells, such as U87(glioma), PC-3(prostate cancer), MDA-MB-468(breast cancer) and Huh-7(liver cancer). In B-cell lymphoma, compared with the CC-223, GT0486 shows greater inhibitory potency against TMD8, DoHH2 and WSU-DLCL2, with IC50 values of 85nM, 96nM and 83nM, respectively. Moreover, in the U87 and the PC-3 xenograft animal models, GT0486 strongly reduced tumor growth in dose dependent manner. High synergistic effects were observed with GT0486 in combination with BTK inhibitors, including Acalabrutinib, Ibrutinib, Zanubrutinib and Orelabrutinib in the BTKi sensitive TMD8 cells. In BTKi resistant DoHH2 cells, GT0486 also enhanced the drug sensitivity of BTKi. The possible biomarkers on the synergistic mechanism were further studied. GT0486 improves the role of BTKi on cell cycle arrest in phase G1. At the same time, the phosphorylation levels of mTOR downstream effectors AKT, 4EBP1 and S6 were dose-dependently downregulated after GT0486 treatment, and a significant reduction were observed when combined with BTKi. These results demonstrate that GT0486 is a promising dual mTOR1/2 inhibitor, which is currently undergoing clinical phase I study in China for the treatment of solid tumors. Strong synergistic effects observed with GT0486 in combination with BTKi in this study might open a way for a novel treatment strategy in clinical trials. Citation Format: Xiaodan Hou, Honghua Yan, Xin Zhang, Weidong Qian, Dong Chen, Zhihua Ren, Youzhi Tong. GT0486, a novel mTORC1/2 dual inhibitor, exhibits synergistic antitumor efficacy in combination with BTK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6509.

Abstract 5842: An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia

Abstract Therapeutic inhibition of BCR signaling using Bruton tyrosine kinase inhibitors (BTKis) has remarkable efficacy in the treatment of chronic lymphocytic leukemia (CLL). However, the efficacy of BTKis is limited by the development of drug resistance, which can be mediated through mutations in genes encoding proteins of BCR signaling pathways. Approaches targeting the BCR mutant proteins have been limited by the large heterogeneity of these mutations. In addition, BCR signaling mutations are only detected in up to two-thirds of the CLL patients who progressed with BTKi treatment, suggesting mechanisms beyond genetic mutations. We previously showed that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells (Wang et al., Blood Cancer J. 2022), suggesting that epigenetic mechanisms are exploited by CLL B cells to support their survival. To gain insights into the epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome-wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on BTKi ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in a CLL cohort at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies showed that suppressing BCR signaling by BTKi leads to the downregulation of genes associated with mitochondrial translation, a key process of mitochondrial biogenesis. We also found decreased chromatin accessibility and H3K27ac deposition at the promoters of these MT genes. Interestingly, in patients who experienced disease progression on ibrutinib, there was a restoration of MT gene expression, chromatin accessibility, and H3K27ac levels at the MT gene promoters similar to baseline samples, suggesting an epigenetic-regulated MT both in BTKi treatment naïve and relapsed CLL patients. Importantly, MT inhibition led to a decreased survival of primary CLL B cells. These findings point toward an epigenetic mechanism utilized by BCR signaling to promote MT and leukemic cell survival. We then observed that the transcription factor NRF1 is enriched at the MT promoter in CLL B cells. CRISPR-mediated depletion of NRF1 led to reduced mitochondrial translation and cell survival in BCR signaling active malignant B cell lines and primary CLL B cells. These results suggest that NRF1 controls an epigenetic program linking BCR signaling to control MT, which in turn supports BCR signaling-dependent CLL B cell survival. Our findings demonstrated that BCR signaling utilizes epigenetic mechanisms that regulate mitochondrial metabolism to enhance cell survival in both treatment naïve and BTKi-relapsed CLL. These findings may provide new therapeutic directions for untreated and BTKi-relapsed CLL patients. Citation Format: Olivia M. Depies, Sutapa Sinha, Sameer Parikh, Neil Kay, Zhiquan Wang. An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5842.

Abstract 57: Ibrutinib improves chimeric antigen receptor T cell control of leukemia by inhibiting myeloid-derived suppressor cells

Abstract Background: In patients with chronic lymphocytic leukemia (CLL), we and others have shown that the addition of the BTK inhibitor ibrutinib to CART cells increases the rates of durable complete responses. The mechanism for this remains unknown. Lymphodepletion (LD) is considered critical for CART cell therapy success, yet emergency myelopoiesis during recovery from LD may induce a surge of myeloid-derived suppressor cells (MDSC), particularly in patients with advanced cancer. BTK inhibition has been shown to inhibit MDSC function in vitro. We therefore hypothesized that administration of ibrutinib during and following LD could reduce MDSC-mediated suppression of CART cells and thereby improve CART cell outcomes. Methods: To explore the effects of ibrutinib on human MDSCs, we engineered suppressive monocytes through in vitro culture of CD14+ cells in the presence of IL-6 and GM-CSF. We added ibrutinib to the culture on Days 0-4 and compared the ability of the monocytes to inhibit proliferation of human CAR-T cells. To model CLL treatment with CART in immunocompetent mice, we used the Em-TCL1 adaptive transfer model of CLL. Mice were treated with ibrutinib (I), cyclophosphamide and fludarabine (FC), or ibrutinib + FC (FC+I). MDSCs were isolated from these mice for in vitro function studies. In subsequent experiments mice were also treated with syngeneic CART19 with or without FC or FC+I. Results: In vitro generated human MDSC suppressed CART19 function, and this was prevented by pre-treatment of MDSC with ibrutinib. Myeloid cells from the spleens of CLL-bearing mice inhibited syngeneic CART19 function ex vivo regardless of treatment with FC or I alone, but myeloid cells harvested from CLL-bearing mice treated with FC+I lost suppressive function. Finally, mice treated with CART19 after a LD regimen resembling that in our recently reported clinical trial (FC+I) showed a survival advantage over all other groups. Conclusions: BTK inhibition with ibrutinib prevents myeloid cell suppression of CART cells in vitro in human and murine systems. When combined with lymphodepleting chemotherapy (FC), ibrutinib enhances CART cell control of CLL in vivo, possibly by preventing MDSC-mediated suppression of CART cells. Citation Format: Benjamin F. Frost, Olga Shestova, Feng Shen, Chia Sharpe, John C. Byrd, Saar I. Gill. Ibrutinib improves chimeric antigen receptor T cell control of leukemia by inhibiting myeloid-derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 57.

Effect of Combined Oral Contraceptive Pills on Established Biomarkers of Lipid Metabolism: Systematic Review and Meta-Analysis

Background and aims: Bruton tyrosine kinase inhibitors, such as ibrutinib, are medications that are actively used for Chronic Lymphocytic Leukemia (CLL) and several lymphomas. They work by inhibiting Bruton’s tyrosine kinase therefore affecting B cell proliferation. Ibrutinib itself is associated with multiple cardiotoxicities including atrial fibrillation, ventricular arrhythmias, heart failure, and bleeding. There are well-established studies that show the incidence of atrial fibrillation with ibrutinib therapy. However, only a few reports show an association between ibrutinib therapy and advanced heart block. Methods: A systematic review was carried out involving searching in research databases including PubMed using key search terms. This was followed by a meta-analysis that compared cholesterol, high density lipoprotein, low-density lipoprotein and triglycerides in a population taking combined oral contraceptive pills containing ethinyl oestradiol/levonorgestrel at 6 months vs the baseline measurement. Results: Six studies (9 intervention groups), totalling 274 participants were analysed. Hedge’s g for the cholesterol comparison was 0.133 (95% confidence intervals -0.058, 0.324; p=0.173; I2 =74.652), which was not significant. In contrast high density lipoprotein was significantly lower, Hedge’s g -0.546 (95% confidence intervals -0.834, -0.259; p<0.001; I2 =92.546) and lowdensity lipoprotein significantly higher, Hedge’s g 0.248 (95% confidence intervals 0.055, 0.442; p=0.012; I2 =83.116 as were the triglycerides, Hedge’s g 0.667 (95% confidence intervals 0.491, 0.842; p<0.001; I2 =64.627. Conclusion: After 6 months use the combined oral contraceptive pills containing ethinyl oestradiol/levonorgestrel led to a significantly increased concentration of low-density lipoprotein and triglycerides and a significantly lower concentration of high-density lipoprotein. More research is required to investigate whether these changes are associated with an increased risk of cardiovascular disease.

Abstract 6044: EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases

Abstract Heterobifunctional kinase degraders (HKDs) are an important new class of therapeutic modalities, of which the first have recently entered clinical trials. HKDs are small molecules that comprise a kinase-of-interest binding moiety and an E3 ligase binding moiety connected via a spacer. HKDs are excellently suited to target kinases that have a scaffolding function, or that are resistant against conventional inhibitors. A HKD brings a kinase and an E3 ligase in proximity forming a ternary complex leading to ubiquitin-dependent catalytic degradation of the kinase of interest. This ternary complex formation is unique for each kinase which can make HKDs highly specific. To discover novel HKDs, we have developed a platform called Energetically Privileged Ligands (EPriLs), which is based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket with near-covalent affinity, and allows binding to kinases that have acquired known inhibitor resistance mutations. The macrocyclic structure allows attachment of E3 ligase ligands with multiple exit vectors. With the EPriL platform, we have developed novel HKDs for various clinically relevant kinase targets, including Bruton’s Tyrosine Kinase (BTK). BTK inhibitor drugs are used as first line treatment for chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and as second line treatment for mantle cell lymphoma (MCL). Despite their great success in the clinic, many patients on BTK inhibitors relapse eventually, mostly because of the emergence of resistance mutations in the BTK protein. These include BTKC481S, which abrogates binding of covalent drugs, and BTKT474I, BTKL528W and BTKV416L which preclude binding of non-covalent inhibitors like pirtobrutinib. HKDs can combat resistance by degrading such BTK variants, and by removing the scaffold function of BTK. BTK degraders such as NX-5948 and NX-2127 are currently in clinical Phase 1. To show the applicability of the EPriL platform for the discovery of novel BTK degraders, we screened a library of more than 100 EPriL HKDs for biochemical binding to BTK, and tested their propensity to induce degradation of HiBiT-tagged BTKWT, BTKC481S and BTKT474I in HEK293 cell lines. The degrading activity of hits was verified through western blot on the BTK-dependent DLBCL cell line TMD8. The best degraders - based on potency and efficacy - were tested in cell proliferation assays on a panel of TMD8 BTK mutant cell lines. In these lines, endogenous BTK was mutated by CRISPR to BTKC481S, BTKT474I, BTKL528W or BTKV416L. Several BTK degraders had potent (< 20 nM IC50) antiproliferative activity on most TMD8 lines, including difficult-to-target variants such as BTKV416L and BTKL528W. The unique binding mode of the EPriL scaffold thus translates to broad activity on resistance variants, while its selectivity ensures minimal degradation of off-target tyrosine kinases as observed for other degraders. Citation Format: Michelle Muller, Jos de Man, Joost C. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G. van Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6044.